The long term goal of this research is to develop robust and reliable ultrasound strategy for intracellular delivery of desirable agents (e.g. drugs, genes, imaging markers) for biomedical applications such as targeted cancer treatment, molecular imaging, and gene therapy. There exists a widely recognized need to develop methods to achieve targeted delivery of drugs and to improve the methods of gene delivery for gene-based therapy of numerous diseases. As ultrasound exposure is safe and non-invasive, and allows targeted application both temporarily and spatially, ultrasound mediated delivery has the potential to provide an advantageous strategy especially for in vivo clinical applications to overcome the limitations of safety concerns, possibly mutagenesis and immune responses associated with methods such as electroporation and viral transfection. It has been demonstrated that ultrasound application results in enhanced intracellular uptake of chemotherapeutic compounds, genetic materials, and fluorescent dextran molecules, which are normally not permeable through intact cell membrane. The hypothesis is that sonoporation, during which pores form in the cell membrane as the result of ultrasound exposure, allowing entry of extracellular molecules and substances into the cell before resealing. However, despite of the recent progress made in the field, the mechanisms of sonoporation are not completely understood and many problems and challenges remain to improve delivery efficiency and cell survival rate. To achieve our goal of developing optimal ultrasound mediated delivery strategy for ultimate clinical applications, this research focuses on investigating the mechanisms of sonoporation by studying the dynamic processes of sonoporation at both the single cell level and the cellular level based on a large number of statistical events. The specific aims are: 1. To study the sonoporation process and mechanism at the single cell level. To systematically investigate and characterize sonoporation, we will develop and establish an integrated approach including novel application of patch clamp technique, dynamic fluorescent imaging, in addition to various assay methods for post-ultrasound analysis. We plan to a) characterize quantitatively the key aspects of sonoporation (pore formation, duration, and resealing) at the cellular level affected by acoustic parameters (frequency, intensity, exposure protocol) and microbubble contrast agents; b) investigate the effects of Ca2+ on sonoporation dynamics (especially membrane resealing) and post ultrasound cell survival; 2. To develop ultrasound method to achieve optimal intracellular delivery outcome. We will investigate and optimize ultrasound intracellular delivery of agents in various relevant formulations of interest (e.g. DNA, fluorescent probes, cancer drugs, and nano-particles) using two model systems: a colonic cancer cell line and adult cardiac myocytes. We will a) establish correlation of delivery outcome (intracellular uptake and cell survival) with sonoporation conditions (ultrasound parameters, Optison concentration, and Calcium); b) achieve optimal delivery outcome through controlling the sonoporation conditions.